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Introduction: Gel Chromatography

  • Gel Chromatography, also known as Size Exclusion Chromatography (SEC), is a technique that separates molecules based on size.

  • It is widely used in the pharmaceutical industry for analyzing and purifying biomolecules such as proteins, peptides, nucleic acids, and polysaccharides.

  • Column Composition: Packed with porous beads made of materials like agarose, dextran, or polyacrylamide.

  • Separation Mechanism: Smaller molecules enter the bead pores, eluting slower; larger molecules bypass the pores and elute faster.

Introduction: Gel Chromatography
Introduction: Gel Chromatography

Principle

  • Gel Chromatography operates on the size exclusion principle, where separation depends on a molecule's size or hydrodynamic volume:

  • Large Molecules: Cannot enter the pores, elute quickly.

  • Intermediate Molecules: Partially enter pores, elute at intermediate times.

  • Small Molecules: Enter most or all pores, elute last.

  • Separation is based on molecular size in solution, not intrinsic size.

Classification of Gel Chromatography

Based on Material and Use:

  • Gel Filtration Chromatography (GFC): For aqueous samples; uses hydrophilic gels (e.g., dextran, agarose); applied in protein purification.

  • Gel Permeation Chromatography (GPC): For synthetic polymers in organic solvents; uses hydrophobic beads (e.g., polystyrene).

Based on Pore Size:

  • Micro-Porous: For low molecular weight compounds.

  • Meso-Porous: For medium molecular weight compounds.

  • Macro-Porous: For high molecular weight compounds.

Based on Usage:

  • Analytical Chromatography: Analyzes molecular weight, size, and purity.

  • Preparative Chromatography: Purifies and collects specific molecules in large quantities.

Advantages:

  1. Mild Conditions: Works under non-denaturing conditions, preserving the biological activity of sensitive molecules like enzymes or proteins.

  2. Versatility: Can be used for a wide range of molecules from small ions to large proteins or nucleic acids.

  3. High Resolution: Can effectively separate molecules with minor size differences.

  4. Desalting Capability: Efficient in removing small molecules like salts from larger ones in a sample.

Disadvantages:

  1. Limited Range: Each column has a specific molecular weight range. Molecules outside this range might not be separated efficiently.

  2. Bead Degradation: Over time, or under harsh conditions, the beads can degrade or shrink.

  3. Limited Chemical Compatibility: Some gels can be incompatible with certain solvents or conditions.

  4. Sample Recovery: Sometimes, the sample might interact non-specifically with the matrix, leading to reduced recovery.


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